Characterisation of naturally occurring Erwinia amylovora strains lacking the common plasmid pEA29 and their detection with real-time PCR

Abstract

Erwinia amylovora, the causal agent of fire blight, carries the common plasmid pEA29 of 29 kb. To screen for occurrence of natural strains without plasmid pEA29, we applied PCR analysis with primers from the plasmid and the chromosomal ams region. In addition, a described TaqMan probe from pEA29 and newly designed primers from the ams-region were used for identification by real-time PCR. One strain isolated in Iran, one strain from Spain and two strains from Egypt lacked plasmid pEA29. From a recent screening series in southern Germany, in 123 E. amylovora strains from necrotic fire blight host plants, one strain was found without the common plasmid. The strains without pEA29 were virulent in assays with immature pears and on apple seedlings, but showed a reduced growth level in minimal medium without amino acids and thiamine. Transposon-labelled pEA29 was transformed into the plasmid-free strains resulting in restoration of this growth deficiency. The plasmid was stably maintained in these E. amylovora cells. The newly designed chromosomal primers for conventional and for real-time PCR identified E. amylovora strains in field samples lacking pEA29. These variants are apparently rare, but were detected in isolates from different regions in the world with fire blight.